Abstract

In normal physiologic responses to injury and infection, inflammatory cells enter tissue and sites of inflammation through a chemotactic process regulated by several families of proteins, including inflammatory chemokines, a family of small inducible cytokines. In neutrophils, chemokines chemokine (CXC motif) ligand 1 (CXCL1) and CXCL8 are potent chemoattractants and activate G protein–coupled receptors CXC chemokine receptor 1 (CXCR1) and CXCR2. Several small-molecule antagonists of CXCR2 have been developed to inhibit the inflammatory responses mediated by this receptor. Here, we present the data describing the pharmacology of AZD5069 [N-(2-(2,3-difluorobenzylthio)-6-((2R,3S)-3,4-dihydroxybutan-2-yloxy)[2,4,5,6-13C4, 1,3-15N2]pyrimidin-4-yl)azetidine-1-sulfonamide,[15N2,13C4]N-(2-(2,3-difluoro-6-[3H]-benzylthio)-6-((2R,3S)-3,4-dihydroxybutan-2-yloxy)pyrimidin-4-yl)azetidine-1-sulfonamide], a novel antagonist of CXCR2. AZD5069 was shown to inhibit binding of radiolabeled CXCL8 to human CXCR2 with a pIC50 value of 9.1. Furthermore, AZD5069 inhibited neutrophil chemotaxis, with a pA2 of approximately 9.6, and adhesion molecule expression, with a pA2 of 6.9, in response to CXCL1. AZD5069 was a slowly reversible antagonist of CXCR2 with effects of time and temperature evident on the pharmacology and binding kinetics. With short incubation times, AZD5069 appeared to have an antagonist profile with insurmountable antagonism of calcium response curves. This behavior was also observed in vivo in an acute lipopolysaccharide-induced lung inflammation model. Altogether, the data presented here show that AZD5069 represents a novel, potent, and selective CXCR2 antagonist with potential as a therapeutic agent in inflammatory conditions.

Introduction

Overstimulation of the inflammatory response plays a role in the pathology of a variety of diseases. The process is propagated by circulating leukocytes entering into inflamed tissue in response to inflammatory mediators. The inflammatory cells entering the tissue are directed through a chemotactic process regulated by several families of proteins, including inflammatory cytokines, adhesion molecules, matrix metalloproteases, and chemokines.

The CXC chemokine receptor 2 (CXCR2) is a G protein–coupled receptor for a number of chemokines and is known to be elevated in several inflammatory diseases, including chronic obstructive pulmonary disease (COPD), severe asthma, acute respiratory distress syndrome, rheumatoid arthritis, psoriasis, and inflammatory bowel disease. In addition, both CXCL8 and CXCL1 (endogenous ligands of CXCR2) have been shown to be increased in inflammation (Schulz et al., 1993; Kurdowska et al., 2002; Banks et al., 2003; Beeh et al., 2003). Recent advances indicate that chronic inflammation is a key risk factor for cancer, and there are several associations between CXCR2 and cancer (Jamieson et al., 2012; Katoh et al., 2013; Highfill et al., 2014).

Chemokines are important in host defense mechanisms such as calcium mobilization, release of granule contents, and respiratory burst. Chemokine CXCL8 activates both CXCR1 and CXCR2. CXCR1 is stimulated by relatively few chemokines, CXCL6, and possibly CXCL7, whereas CXCR2 binds to all seven ELR+ (glutamate-leucine-arginine ELR tripeptide motif) CXC chemokines, CXCL1–CXCL3 and CXCL5–CXCL8 (Bachelerie et al., 2014). These are potent chemoattractants for neutrophils, and hence, CXCR1 and CXCR2 constitute the primary mechanism for recruitment of neutrophils to sites of inflammation and infection. CXCR1 and CXCR2 have also been detected on other cells associated with chronic inflammation, including macrophages, lymphocytes, mast cells, dendritic cells, and endothelial cells (Murdoch and Finn, 2000; Mukaida et al., 2003; Traves et al., 2004; Reutershan et al., 2006).

CXCR1 and CXCR2 have similar signaling mechanisms (Stillie et al., 2009), and CXCL8 can potentiate several neutrophil functions triggered through both receptors, including phosphoinositide hydrolysis, intracellular Ca2+ mobilization, and chemotaxis. However, CXCR1 has been specifically implicated in phospholipase D activation, respiratory burst activity, and the bacterial-killing capacity of neutrophils (Jones et al., 1996), suggesting that CXCR1 and CXCR2 might have different physiologic roles under inflammatory conditions.

Several small-molecule chemokine receptor antagonists have been developed as a potential therapeutic approach for the treatment of inflammatory disease—for example, repertaxin, navarixin, and danirixin (Chapman et al., 2009). These are in different stages of drug development and have been shown to have an effect on neutrophil recruitment to the lung in clinical studies (Holz et al., 2010; Lazaar et al., 2011; Virtala et al., 2012; Pavord et al., 2013). In addition, the effect of inhibiting neutrophil recruitment has been shown for clinical biomarkers and endpoints indicative of disease efficacy as investigated in cystic fibrosis, severe asthma, and COPD (Nair et al., 2012; Moss et al., 2013; Rennard et al., 2015). Despite a strong association of chemokine involvement in disease, to date, there are only two marketed products targeting chemokine receptors: plerixafor, a small-molecule antagonist of CXCR4, and maraviroc, an antagonist of CCR5 (Bachelerie et al., 2014).

AZD5069 [N-(2-(2,3-difluorobenzylthio)-6-((2R,3S)-3,4-dihydroxybutan-2-yloxy)[2,4,5,6-13C4, 1,3-15N2]pyrimidin-4-yl)azetidine-1-sulfonamide,[15N2,13C4]N-(2-(2,3-difluoro-6-[3H]-benzylthio)-6-((2R,3S)-3,4-dihydroxybutan-2-yloxy)pyrimidin-4-yl)azetidine-1-sulfonamide] was developed from a series of bicyclic CXCR2 antagonists (Walters et al., 2008; Allen et al., 2014). The studies presented here describe the pharmacological characterization of AZD5069 as a potent, reversible antagonist of CXCR2 and the effect in a rat in vivo lipopolysaccharide (LPS) challenge model of lung inflammation. These data are discussed in the context of the properties of this compound determined in vitro. AZD5069 represents a novel small-molecule antagonist for the potential treatment of inflammatory conditions arising from neutrophil infiltration.

Cell Membranes.

Cells were resuspended on ice in hypotonic buffer and disrupted using a polytron tissue homogenizer at 22,000 rpm. The membrane preparation was purified by layering onto 41% (w/v) sucrose solution, and then centrifuged at 140,000g for 1 hour at 4°C. The membrane fraction at the interface was harvested, diluted 4-fold with HEPES-buffered salt solution (pH 7.4), and centrifuged at 100,000g for 20 minutes at 4°C. The membrane pellet was resuspended in HEPES-buffered salt solution and subsequently stored in aliquots at −80°C. All buffers used for membrane preparation and storage were made in the presence of complete protease inhibitor cocktail tablets (Sigma-Aldrich, St. Louis, MO), prepared according to the manufacturer’s instructions.

Polymorphonuclear Cell Preparation.

Peripheral venous human blood was collected in polypropylene tubes containing 10 U/ml of heparin, then 25 ml of blood was layered onto 17 ml of PolymorphPrep (Robbins Scientific, Sunnyvale, CA) and centrifuged at 450g for 30 minutes at room temperature. The polymorphonuclear (PMN) layer was placed into a fresh polypropylene centrifuge tube and diluted in an equal volume of phosphate-buffered saline (PBS) containing 0.2% (w/v) glucose. The cells were centrifuged at 300g for 5 minutes at room temperature. The supernatant was decanted and the cell pellet dispersed. Hypotonic lysis of red blood cells was performed by resuspending the pellet in 7.5 ml of 0.2% (w/v) NaCl for 1 minute while inverting the tube and then adding an equal volume of 1.6% (w/v) NaCl. PBS (35 ml) containing 0.2% (w/v) glucose was added, and the cells were centrifuged at 300g for 5 minutes. The procedure was repeated to ensure complete lysis of red blood cells. The PMN cell pellet was resuspended in 20 ml of Tyrode’s/HEPES solution and the cell number determined.

CXCL8 Radioligand Binding.

Radiolabeled [125I]CXCL8 was used in the assay at a final concentration of 0.06 nM. The assay was performed in 0.1% dimethylsulfoxide (DMSO), and nonspecific binding was determined in the presence of 1 µM AZ10397767. The assay was initiated by adding membranes to give a total incubation volume of 100 μl per well in a 96-well multiscreen filter plate, prewetted with assay buffer. The plates were sealed and incubated for 2 hours at 22°C. Plates containing the assay mixture were filtered and then filter washed with 200 µl of cold HEPES-buffered salt solution using a Millipore (Billerica, MA) vacuum manifold. The filtration plate was allowed to air dry, then the individual filters were punched out into polypropylene test tubes and the radioactivity measured by direct gamma counting using a Cobra II gamma counter (Packard BioScience, Meriden, CT) for 1 minute per sample. Data were analyzed using 4-parameter logistic function in the Excel-based program XL-fit (I.D. Business Solutions Ltd., Guildford, UK).

AZD5069 Radioligand Binding.

Radiolabeled [3H]AZD5069 was prepared at AstraZeneca R&D Charnwood. The affinity of AZD5069 was quantified by determining the specific binding of [3H]AZD5069 to membranes of HEK293 cells expressing recombinant CXCR2 from various species. Nonspecific binding of [3H]AZD5069 was determined in the presence of 10 μM AZ11705135 [N-(3-aminosulphonyl-4-chloro-2-hydroxy)-N′-(2,3-dichlorophenyl)urea], a concentration much higher than the IC50 of this compound (2 nM), to displace radiolabeled CXCL8.

Membranes from HEK293 cells expressing recombinant CXCR2 were suspended in HEPES-buffered salt solution. The membranes were then bound to wheat germ agglutinin scintillation proximity assay (SPA) beads by mixing 37-μl SPA beads (100 mg/ml in water) with 10 ml of diluted membrane, and incubating at 37°C for 1 hour with gentle agitation. Membrane-bound beads were then centrifuged for 5 minutes at 1000 rpm. The supernatant was drained off and replaced with fresh HEPES-buffered salt solution. [3H]AZD5069 was added to each well of a white clear-bottomed 96-well plate in the presence and absence of 10 μM AZ11705135 to determine nonspecific binding. The binding of [3H]AZD5069 was initiated by the addition of SPA bead-membrane preincubation mix to the wells in a total assay volume of 200 μl. The plate was sealed, mixed, and incubated at 37°C for 2 hours. Radioactivity was determined in a TriLux 1450 Microbeta Scintillation counter (PerkinElmer, Coventry, UK) with a 1-minute read time. The nonspecific binding control was subtracted, then the data were fitted to a one-site binding hyperbola using the GraphPad Prism Software package (GraphPad Software, La Jolla, CA).

HEK Calcium Flux Assay.

HEK293 cells, expressing recombinant human CXCR2 and Gqi5, were grown and harvested as described earlier. The assay was performed in 96-well poly-d-lysine–coated plates (Becton Dickinson, Franklin Lakes, NJ). To each well, 25,000 cells were added in a final volume of 100 μl. Cells were allowed to adhere to the plates overnight at 37°C. Adhered cells were washed twice in buffer and test compound solution containing 1.5% (v/v) DMSO, and were added to the appropriate wells at 3× final concentration in a volume of 50 μl. The cells were incubated with compound at either 22°C for 30 minutes or 37°C for 3 hours. Prior to measurement of intracellular calcium transients, cells were preloaded with Fluo-4 AM solution for 60 minutes. Calcium levels were monitored using a fluorescence imaging plate reader (Molecular Devices, Sunnyvale, CA) after 50 μl of solutions containing CXCL8 at 4× final concentration had been added. Calcium transients in response to various concentrations of CXCL8 were measured. Readings were taken every 2 seconds for a total run time of 6 minutes.

Polymorphonuclear Cell Calcium Flux Assay.

One hundred microliters of Fluo-3 AM solution diluted to 5 μM final concentration was added and the PMN cell suspension rolled for 90 minutes at room temperature. Fluo-3 AM–loaded cells were centrifuged at 300g for 5 minutes at room temperature, the supernatant was discarded, then the cells were resuspended in Tyrode’s/HEPES solution at a concentration of 4 × 106/ml.

The assay was performed in 96-well poly-d-lysine–coated plates (Becton Dickinson). To each well, 200,000 cells and test compound in 100 μl of solution was added. Assay buffer containing 1.5% (v/v) DMSO and compound at 3× final concentration was added to the appropriate wells. The 96-well plates were centrifuged (200g, 5 minutes) and then incubated at room temperature for 30 minutes in 0.75% DMSO. Calcium transients in response to various concentrations of CXCL1 were measured using a fluorescence imaging plate reader, where 50 μl of solutions containing CXCL1 at 3× final concentration was added and fluorescence readings were taken every 2 seconds for a total run time of 2 minutes. The CXCR2 selective ligand CXCL1 was used in PMN cells to investigate CXCR2-mediated effects in these cells as they express both CXCR2 and CXCR1 receptors.

Reversibility of Calcium Flux Inhibition in HEK Cells.

HEK293 cells, expressing recombinant human CXCR2 and Gqi5, were grown and harvested as described earlier. Cells and plates were set up as described earlier. AZD5069 solution or PBS/HEPES solution, containing 1.5% (v/v) DMSO, was added to the appropriate wells in a volume of 50 μl to give a final concentration of 3 nM. Cells were incubated with vehicle or compound at 37°C for 2 hours and then washed three times using 100 μl of buffer and left in 100 μl of buffer for 4 hours before reading. A time-matched control was also conducted where cells were incubated in compound for 2 hours, 4 hours after the start of the experiment with no wash before reading. One hour before reading, 50 μl of Fluo-4 AM solution was added and the cells were incubated for a further 60 minutes at 37°C. Calcium transients were then measured as described previously.

Chemotaxis.

PMN cells were prepared as described earlier. A chemotactic gradient to CXCL1 in the presence and absence of test compound was performed in 96-well Neuroprobe chemotaxis plates (Receptor Technologies, Warwick, UK) with a 3-μm filter. An equal volume of compound or vehicle control was added to cells in Tyrode’s/HEPES solution. Cells in the presence or absence of compound were then incubated while rolling for 30 minutes at room temperature. Solutions of CXCL1 and compound were added to the lower well compartment of the chemotaxis plate. A 25-μl droplet of isolated PMN cells at 8 × 106/ml was placed on top of each filter position. The filter plate assembly was incubated at 37°C in 5% CO2 for 3 hours to provide sufficient time for a slow-acting compound to demonstrate an effect. The filter was removed and 5 μl of Alamar Blue was added to the cells in the lower well compartment. Cells were incubated at 37°C in 5% CO2 for 45 minutes and the fluorescence determined at 560 nm using an FMAX fluorimeter (Molecular Devices). Changes in fluorescence were expressed as the number of cells by comparison with a calibration performed with Alamar Blue. The number of cells responding to the chemotactic gradient was expressed as a percentage relative to the maximum effect observed in the vehicle control.

Whole-Blood CD11b Expression.

Blood was collected in lithium heparin–coated (final concentration 10 IU/ml) tubes. Within 20 minute of collection, 2.7 μl of DMSO vehicle control or various concentrations of compounds in DMSO were added to individual tubes of blood to give a final DMSO concentration of 0.03%. Each tube was rolled gently at room temperature for 60 minutes. Aliquots (80 μl) of the blood incubations were then added to deep-well polypropylene 96-well plates containing fluorescein isothiocyanate (FITC)–labeled CD11b and phycoerythrin-labeled CD16 antibodies or isotype controls (Caltag Medsystems Ltd., Buckingham, UK). Cells were stimulated by addition of various concentrations of agonist (10 μl) mixed for 5 seconds, then incubated at room temperature in the dark for 40 minutes. The assay was stopped by placing cells on ice for 30 minutes. Plates were removed from the ice bath and fixed in Optilyse B (Beckman Coulter (UK) Ltd, High Wycombe, UK) by adding 0.1 m Optilyse B followed by vigorous shaking for 10 minutes at room temperature. Distilled water (1 ml) was added to each well and the plate was mixed for at least 30 minutes at room temperature. Samples were then transferred to polypropylene LP4 tubes and stored in the dark at 4°C until ready to analyze by flow cytometry. CD11b expression was determined using a Coulter XL flow cytometer (Beckman Coulter (UK) Ltd) to detect the level of FITC fluorescence on the CD16-positive granular cells with high forward and side-scatter properties. Within the granulocyte region, cells with high levels of CD16-PE fluorescence were gated as the “neutrophil region.” Neutrophil CD11b expression on the CD16-positive neutrophil population was measured by determining the median FITC fluorescence. Data describing concentration effect curves were fitted to a 4-parameter logistic function using “XL-fit,” and the response was expressed as a percentage relative to the maximum agonist effect observed in the vehicle control.

AZD5069 Radioligand Binding Kinetics.

The dissociation for AZD5069 was measured by displacement of [3H]AZD5069 from membranes of HEK293 cells expressing recombinant human CXCR2. Membranes from HEK cells expressing CXCR2 were bound to wheat germ hemagglutinin SPA beads as described earlier. [3H]AZD5069 at various concentrations and either buffer or AZ10397767 (10 μM final concentration) was added to each well of a white clear-bottomed 96-well plate to determine the nonspecific binding. The binding of [3H]AZD5069 was initiated by the addition of 160 μl of membrane-coated SPA beads to the wells. The plate was sealed, mixed, and incubated at either 37°C for 90 minutes or 22°C for 200 minutes. The dissociation of [3H]AZD5069 was initiated by the addition of AZ10397767 to 10 μM in buffer containing 0.1% DMSO. The plate was mixed and counts per minute were sequentially determined at various time intervals in a Trilux 1450 Microbeta Scintillation counter (Wallac Ltd.). For experiments performed at 22°C, data points were collected in real time by incubation within the scintillation counter. For experiments incubated at 37°C, plates were removed from the incubator briefly for scintillation counting for 30 seconds before being returned to the incubator. The nonspecific binding control data were subtracted from the measured counts per minute, and the data were fitted to a single exponential decay model using the Origin Software package (OriginLab, Northampton, MA).

Rat Airway LPS Challenge Model.

All animal studies were conducted under an approved UK Home Office project license. The compound doses were selected based on a combination of data from in vitro CXCR2 potency, rat pharmacokinetics, and rat protein binding. Male CRL:CD rats (weight range 315–427 g) were allowed to acclimatize to housing conditions for at least 7 days after delivery. Compounds were prepared in vehicle (1% hydroxypropyl methylcellulose/0.1% Tween 80/H2O).

Rats in treatment groups (eight per group) were dosed orally 1 hour before LPS challenge with AZD5069, AZD8309, or AZ10397767 or dexamethasone (5.8 μmol/kg). Rats in group 1 were challenged with 0.9% saline. Rats in groups were challenged with 0.1 mg/ml LPS in saline (0.9%) or saline vehicle control. The rats were placed in perspex boxes and challenged with an aerosol generated with two jet nebulizers operated at an airflow rate of 12 l/min for 30 minutes. At 4 hours following LPS challenge, the trachea was cannulated and the airway lavaged using 3 aliquots of 3.3 ml of sterile PBS at room temperature. An aliquot of lavage fluid was removed for cell counting. Cytospin slides were prepared by adding a 100-μl aliquot of lavage fluid into cytospin funnels in a Shandon Cytospin3 (GMI, Inc., Ramsey, MN) operated at 700 rpm for 5 minutes. Slides were stained with Wright-Giemsa stain using a Hema-Tek-2000 automatic slide stainer (Fisher Scientific Ltd, UK, Loughborough, UK), and typically, 200 cells were counted under a microscope. Cells were classified as eosinophils, neutrophils, and mononuclear cells. Mononuclear cells included monocytes, macrophages, and lymphocytes. The number of neutrophils was quantified by expressing the cell number as a percentage of the total count. The number of neutrophils per animal was averaged across each treatment group and the result expressed as the mean ± S.E.M. Results between treatment groups were compared using nonparametric statistics, Mann-Whitney, or Kruskal-Wallis methodology in GraphPad InStat. Rats were anesthetized with isofluorane, and terminal blood samples (0.5 and 2 ml) were taken from the abdominal vena cava of animals and collected. Animals were then terminated with 1.0 ml of pentobarbitone sodium intraperitoneally. One set of blood samples was assessed for differential cell numbers using an Advia Haematology System (Siemens, London, UK). The other set of blood samples (2 ml) was centrifuged at 2800g for 10 minutes at 4°C. The plasma was removed and stored at −20°C for subsequent determination of compound concentration.

Results

Inhibition of CXCL8 Binding and Specificity.

Initial studies showed AZD5069 inhibited binding of radiolabeled CXCL8 to recombinant membranes expressing human CXCR2 and CXCR1. This system specifically measured binding to the recombinant receptor, as there was no significant binding to HEK293 cell membranes not expressing these receptors. AZD5069, AZD8309, and AZ10397767 all inhibited CXCL8 binding to CXCR2 to a similar degree, with pIC50 values ± S.E.M. of 9.1 ± 0.05, 9.0 ± 0.04, and 8.8 ± 0.06, respectively (Fig. 2). All three compounds also inhibited CXCL8 binding to CXCR1 to a similar degree, with pIC50 values of 6.9 ± 0.08, 6.6 ± 0.10, and 6.5 ± 0.06, respectively. AZD5069 was investigated at 153 human receptor and enzyme targets at a concentration of 10 µM or higher. Significant activity was observed only at two of these targets: CCR2 (pEC50 = 5.2) and CCR5 (pEC50 = 4.9) (Supplemental Tables 1 and 2).

Cross-Species Activity of AZD5069.

The specific binding of [3H]AZD5069 to HEK cell recombinant membranes was used to quantify the binding affinity of AZD5069 to CXCR2 across different species (Fig. 3). pKD values ± S.E.M. for [3H]AZD5069 binding to human, cynomolgus monkey, rat, and dog were determined to be 9.4 ± 0.11, 9.3 ± 0.06, 9.2 ± 0.05, and 9.0 ± 0.17 respectively.

Calcium Responses in Recombinant HEK Cells and PMNs.

An increase in intracellular calcium was observed either when recombinant cells expressing CXCR2 were stimulated with CXCL8 or donor isolated PMNs were stimulated with CXCL1 (Fig. 4, A and D). AZD5069 produced a clear concentration-dependent inhibition of calcium responses in both cell types when AZD5069 was preincubated with cells for a relatively short time (30 minutes) and at room temperature. The pharmacological profile showed either little or no effect at low concentrations of AZD5069 or marked inhibition and curve collapse at high concentrations of AZD5069. Due to the nonclassic agonist responses in the presence of AZD5069, an estimate of pA2 could not be determined under these conditions. However, qualitatively, it can be seen that AZD5069 more potently inhibited calcium responses in PMN cells than in HEK cells expressing recombinant CXCR2. In contrast, the profiles for agonist response curves in the presence of AZD8309 and AZ10397767 shifted in parallel with increasing concentrations of compound for both cell types (Fig. 4, B, C, E, and F). For AZD8309 and AZ10397767, pA2 ± S.E.M. values were determined to be 9.0 ± 0.11 and 8.7 ± 0.08, respectively, for CXCL8-stimulated calcium responses from HEK cells, with more potent inhibition observed for CXCL1-stimulated calcium responses from PMN cells (pA2 ± S.E.M. determined to be 10.0 ± 0.1 and 9.5 ± 0.1 for HEK and PMN cells, respectively).

Effect of compounds on intracellular calcium responses in HEK293 and PMN cells at 22°C following preincubation with compounds for 30 minutes. (A–C) Concentration response curves for CXCL8-induced calcium flux in HEK293 cells expressing human CXCR2 in the presence and absence of compounds. (D–F) Concentration response curves for CXCL1-induced calcium flux in human PMN cells. Compound concentrations were 10 (solid circles), 32 (open triangles), and 100 nM (solid triangles), with the control response in the absence of compound denoted by open circles. Values are the mean ± S.E.M. (n = 6–8).

Effect of Time and Temperature on the Pharmacology and Reversibility of AZD5069.

An assessment was made to determine whether the nonclassic pharmacology of AZD5069 persisted under physiologically relevant conditions of prolonged exposure at 37°C. When AZD5069 was preincubated with HEK cells expressing recombinant CXCR2 for 3 hours at 37°C, the pharmacological profile of the agonist curves demonstrated an apparent parallel shift in agonist curves with increasing concentrations of AZD5069. With the increased time and temperature of incubation, there was also a more regular pattern to the response curves, with an intermediate level of inhibition evident (Fig. 5A) as opposed to only low or high levels of inhibition observed during short preincubation at room temperature (Fig. 4A). Under conditions of 3-hour preincubation at 37°C, a pA2 ± S.E.M. was determined across seven experiments to be 9.6 ± 0.11. However, for eight other experiments, a pA2 could not be determined due to excessive curve collapse. The data shown in Fig. 5A are a composite from all 15 experiments. Prolonged incubation at 37°C could not be performed in PMN cells due to loss of function under these conditions. The reversibility of AZD5069 as an inhibitor of a human CXCR2-mediated response was determined in vitro by measuring inhibition of CXCL8-induced calcium flux in HEK293 cells expressing recombinant human CXCR2 and Gqi5. The reversibility of the inhibitory effect of AZD5069 was demonstrated by washing compound from the cells followed by a 4-hour recovery period. AZD5069 (3 nM) produced a concentration-dependent inhibition of calcium flux induced by CXCL8 (Fig. 5B). The inhibition of functional responses to CXCL8 by AZD5069 was reversible after compound was removed by washing cells. Although the washing procedure used to remove compound from the system was able to demonstrate that inhibition by AZD5069 was reversible, complete restoration of the CXCL8-mediated calcium response to control levels was not observed.

(A) Concentration response curves for CXCL8-induced calcium flux in HEK293 cells expressing human CXCR2 in the presence and absence of AZD5069. Cells were incubated at 37°C for 2 hours. After 1 hour, the Fluo-4 AM solution was added and the cells incubated for a further 60 minutes at 37°C. Compound concentrations were 0.32 (solid squares), 1 (open diamonds), and 3.2 nM (solid diamonds), with the control response in the absence of compound denoted by open circles. Values are the mean ± S.E.M. (n = 15). (B) Concentration response curves for CXCL8-induced calcium flux in HEK293 cells expressing human CXCR2 in the presence (triangles) and absence (circles) of AZD5069. Solid symbols denote initial control data; open symbols denote data collected 4 hours following a washing procedure to remove compound. Values are the mean ± S.E.M. (n = 5).

Inhibition of Agonist-Mediated CD11b Expression and Chemotaxis in Neutrophils and PMNs.

AZD5069 produced a concentration-dependent inhibition of PMN chemotaxis induced by CXCL1 (Fig. 6A). Agonist response curves appeared to collapse in the presence of AZD5069. In contrast, less curve collapse was evident at higher concentrations of the CXCL1-driven PMN chemotaxis response in the presence of AZD8309 or AZ10397767. Although response to a gradient of chemokine is not a simple equilibrium-driven system, the pA2 for AZD5069 was estimated to be approximately 9.6 (range 9.2–9.9). The integrin CD11b is upregulated on neutrophils in response to a variety of inflammatory mediators. AZD5069 produced clear inhibition of CXCL1-mediated CD11b expression with a rightward shift of the concentration effect curve to CXCL1 (Fig. 6B), with a pA2 of 6.9 ± 0.13 (mean ± S.E.M., n = 8). CXCL1 agonist response curves also appeared to collapse in the presence of AZD5069 (Fig. 6B). AZD8309 and AZ10397767 demonstrated inhibition of CXCL1-mediated CD11b expression with a parallel shift in the agonist responses and no apparent decrease in maximum effect (Fig. 6, B and C). This equated to pA2 values of 6.2 ± 0.10 (mean ± S.E.M., n = 6) and 5.8 ± 0.09 (mean ± S.E.M., n = 15) for AZD8309 and AZ10397767, respectively. When several agonists inducing CD11b expression on neutrophils were investigated, a high concentration of AZD5069 was found to specifically inhibit only the CXCL1-driven response with no significant inhibition observed for neutrophils stimulated by C5a, N-formyl-methionyl-leucyl-phenylalanine, or leukotriene B4 (Fig. 7).

Concentration response curves for CXCL1 (A), C5a (B), N-formyl-methionyl-leucyl-phenylalanine (C) and leukotriene B4 (LTB4) (D) stimulating CD11b expression on human neutrophils in blood in the presence and absence of AZD5069. AZD5069 concentrations were 10 μM (solid circles) with the control response in the absence of compound denoted by open circles. Values are the mean ± S.E.M. (n = 4).

Dissociation Kinetics for AZD5069.

Dissociation of [3H]AZD5069 from CXCR2 in the presence of 10 μM AZ10397767 followed a single-site exponential decay model consistent with a simple first-order dissociation process (Fig. 8). Temperature had a marked effect on the dissociation half-life. The dissociation of AZD5069 was shown to be slow at both 37 and 22°C, but changed by more than 12-fold for the 15°C decrease in temperature from a half-life of 28 ± 2 minutes (n = 4) at 37°C to 360 ± 5 minutes (n = 3) at 22°C.

Time dependence for displacement of [3H]AZD5069 (1 nM) with 10 μM AZ10397767 from HEK membranes expressing human CXCR2. Data are expressed as the percentage of specific binding of AZD5069. Circles denote incubation at 37°C, and triangles denote incubation at 22°C. Values are the mean ± S.E.M. (n = 2–4).

AZD5069 Inhibition of LPS-Induced Neutrophilia.

Rat LPS-induced lung neutrophilia is a well validated model of nonallergic airway inflammation (Holmes et al., 2002). LPS inhalation in the rat initiates a cascade of events resulting in pulmonary inflammation characterized by neutrophil influx and associated pathology in the lung. At 4 hours after LPS challenge, there is a robust, reproducible neutrophilia observed in the bronchoalveolar lavage fluid (BALF), which is significantly inhibited by standard anti-inflammatory compounds such as dexamethasone. A dose of 0.1 mg/ml LPS was chosen because it produces a robust and reproducible neutrophil influx that is submaximal and responsive to anti-inflammatory compounds.

LPS challenge significantly increased neutrophil levels in the BALF compared with saline-challenged animals. Orally administered AZD5069 reduced the LPS-induced neutrophilia in a dose-dependent manner (Fig. 9A). The effect of AZD5069 was statistically significant at 3 μmol/kg (P < 0.05) and 10 μmol/kg (P < 0.001), when the terminal plasma concentrations of AZD5069 were 60 and 310 ng/ml, respectively. AZD5069 also inhibited the LPS-induced blood neutrophilia in a dose-dependent manner (Table 1). AZD5069 showed a steep dose response with no effect at 0.3 μmol/kg. In the present study, dexamethasone was used as a positive control and significantly reduced neutrophils in the BALF in all studies. However, in contrast to the steep dose-response relationship observed with AZD5069, both AZD8309 and AZ10397767 exhibited a more gradual dose-response relationship (Fig. 9, B and C).

Dose response for AZD5069 (A), AZD8309 (B), and AZ10397767 (C) on neutrophils recovered from BALF 4 hours after LPS challenge. Dexamethasone dosed at 5.8 µg/kg was used as a control in both studies. Values shown are the mean ± S.E.M. for the number of neutrophils recovered per rat. The statistical significance of compound effects relative to the LPS challenge controls was determined by nonparametric analysis of variance and is denoted as *P < 0.05 and **P < 0.001.

Discussion

We profiled a series of compounds with different structures in the same cell systems to link the translational pathway between human in vitro pharmacology and animal physiology. AZD5069 is a potent, slowly reversible, and selective antagonist of radiolabeled CXCL8 binding at human CXCR2. AZD5069 was developed with the aim of being selective for CXCR2. When measured in a similar system expressing recombinant CXCR1, it was apparent that all of the compounds in this study were around 100-fold more potent at CXCR2 than at CXCR1. This is comparable to known CXCR2 antagonists such as MK-7123 [2-hydroxy-N,N-dimethyl-3-((2-((1(R)-(5-methyl-2-furanyl)propyl)amino)-3,4-dioxo-1-cyclobuten-1-yl)amino)benzamide], which is 80-fold less potent at CXCR1 than CXCR2 (Gonsiorek et al., 2007). Inhibition of CXCR1 function is associated with impaired bacterial killing and could, if specifically inhibited, potentially lead to risks such as increased susceptibility to bacterial infection in humans (Hartl et al., 2007).

Binding of radiolabeled AZD5069 to membranes expressing recombinant CXCR2 was similar for receptor orthologs across four different species: human, cynomolgus monkey, dog, and rat. This is consistent with good cross-species activity demonstrated for other CXCR2 antagonists of different structural classes (Chapman et al., 2007). This cross-species activity of AZD5069 allows the use of AZD5069 as an effective tool compound to explore pharmacological consequences of CXCR2 antagonism in various animal models, as well as the toxicological consequences of both on-target and off-target effects.

AZD5069, AZD8309, and AZ10397767 were all antagonists of CXCL8-mediated increases in intracellular calcium in HEK cells expressing CXCR2. In this system, the pharmacological profiles of AZD8309 and AZ10397767 were consistent with classic competitive antagonism, i.e., parallel rightward shifts of the agonist curves in the presence of increasing concentrations of antagonist, with no apparent significant change in the maximum calcium response at high agonist concentrations (Fig. 4, B and C). In contrast, AZD5069 showed unusual pharmacological behavior with respect to calcium flux antagonism. A small increase in AZD5069 concentration from 32 to 100 nM produced a marked increase in antagonism with significant curve collapse (Fig. 4A).

This profile of antagonism, with curve collapse, was also observed following the inhibition of CXCL1-stimulated calcium release in PMN cells (Fig. 4, D–F). As these data were collected following a 30-minute incubation of compound at 22°C, it was considered that the unusual pharmacological profile of AZD5069 may be due to poor equilibration of AZD5069 with agonist in both the HEK and PMN calcium responses. When the calcium assay using recombinant HEK cells was performed at 37°C for 3 hours, AZD5069 showed increased antagonist potency, and with increasing AZD5069 concentrations, a more regular pattern of response-curve depression was observed (Fig. 5A). Increasing the temperature and time of incubation would be expected to increase the rate of attainment of equilibrium conditions. Under these conditions, a more classic antagonist profile was observed, but with insurmountable antagonism still evident in the calcium response curves. This is consistent with what would be expected for a slowly reversible antagonist where preformed antagonist-receptor complexes are reversible but dissociate so slowly that only part of the receptors can be liberated (Charlton and Vauquelin, 2010). These observations suggest that the nonclassic calcium response curves obtained under conditions of 30-minute incubation at 22°C were due to poor equilibration of the antagonist in the assay system. A prolonged incubation at 37°C was attempted in PMN cells to investigate the effects of the compound under more physiologically relevant conditions; however, these cells did not survive long enough under these conditions for a stable measurement to be recorded.

All CXCR2 antagonists in the current study were found to be potent antagonists of CXCL1-mediated PMN chemotaxis. For CXCL1-mediated CD11b expression, AZD5069 displayed rightward-shifted curves, but with some curve collapse evident at higher concentrations. This curve collapse can be explained by a lack of equilibrium conditions similar to the pharmacology observed in the calcium flux experiments. This pattern of antagonism is similar to the insurmountable antagonism of calcium effects observed at the endothelin receptor by the slowly reversible antagonist Macitentan (Gatfield et al., 2012).

The kinetics of AZD5069 dissociation was measured using radio-labeled AZD5069. At room temperature, the dissociation of AZD5069 was more than 12-fold slower than at 37°C (Fig. 8). This difference is larger than a temperature-dependent change of about 2- to 4-fold, which would be expected for a simple thermodynamically driven dissociation event. The large effect of temperature suggests that dissociation of AZD5069 from the receptor is a more complex process possibly involving cooperativity or multiple steps. This process may involve protein isomerization and/or interaction with membrane lipids. Both AZD5069 and AZ10397767 are similar in structure and would be expected to interact with CXCR2 at the same binding site, but it appears that the interaction is slower and more complex with AZD5069. However, as previously suggested for this class of compounds, the antagonist/receptor interaction occurs at a deeply buried internal binding site (Nicholls et al., 2008) and may contribute to the complexity and rate of the antagonist dissociation from the receptor.

AZD5069 was shown to specifically antagonize CXCL1-mediated adhesion molecule expression in PMN cells without affecting the response to other inflammatory mediators. AZD5069 was therefore specific for the CXCR2 receptor and had no effect on the signal transduction mechanism for adhesion molecule expression. This specificity suggests that AZD5069 would be expected to preserve non–CXCR2-mediated innate immune responses to infection.

The anti-inflammatory effect of AZD5069 is anticipated to be produced by reducing neutrophil migration from the systemic circulation into sites of inflammation, including lung mucosa, with additional effects on neutrophil migration from the bone marrow (Kohler et al., 2011). The response by macrophages and epithelial cells to LPS administered to the rat lung results in the release of chemokines which attract inflammatory cells from the microvasculature (Quinton et al., 2004). AZD5069 was an antagonist of LPS-driven lung neutrophilia in rats (Fig. 9A). The dose-response profile for AZD5069 antagonism of the LPS-driven lung neutrophilia was similar to that observed in the in vitro experiment in which AZD5069 failed to equilibrate and showed a large change in antagonism for a relatively small increase in compound concentration. This suggests that, under these conditions, AZD5069 may also equilibrate slowly in vivo in animal models. In contrast, both AZD8309 and AZ10397767 showed a more predictable dose-response relationship in the LPS-induced rat neutrophil assay. These data suggest that the potency of AZD5069 may be underestimated in acute animal models. In a repeated dosing study with sufficient time for equilibration between tissue and receptor, AZD5069 it could be expected to perform according to the law of mass action, and it could be useful to investigate this compound further in chronic disease, such as COPD, severe asthma, arthritis, inflammatory bowel disease, and cancer.

Acknowledgments

The authors thank Anne Brammall and Iwona Hutchinson for support with in vitro studies, and David Wilkinson for providing [3H]AZD5069.

Authorship Contributions

Participated in research design: Nicholls, Gaw, MacIntosh.

Conducted experiments: Nicholls, Wiley, MacIntosh.

Performed data analysis: Nicholls, Wiley, MacIntosh.

Wrote or contributed to the writing of the manuscript: Nicholls, Wiley, Dainty, Phillips, MacIntosh, Gaw, Kärrman Mårdh.

(2014) International Union of Basic and Clinical Pharmacology. [corrected]. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors. Pharmacol Rev66:1–79.